Facilitation of wound healing with CM101/GBS toxin

ABSTRACT

The method of the present invention provides a means of treating a patient having a wound, especially by minimizing scarring and accelerating wound healing, by administering CM101 or GBS toxin isolated from Group B β-hemolytic Streptococcus bacteria. Types of wounds that may be treated include surface and internal wounds. The method of the present invention also includes administration of CM101 or GBS toxin to surgery patients having tumors in order to facilitate wound healing and minimize the likelihood of tumor progression.

TECHNICAL FIELD

[0001] This invention relates to the facilitation of wound healing inpatients, by minimizing scarring and accelerating healing. Thisinvention also relates to the reduction of wound-related tumorprogression.

BACKGROUND

[0002] The normal process of healing a skin wound that has beensurgically induced or is the result of trauma involves formation of ablood clot and, often, a scab. More particularly, first intention, orprimary healing, generally occurs at clean incisions, whereas secondintention, or secondary healing, occurs where wound edges are far apart.The protein fibrin holds the edges of the skin surrounding the woundtogether and the scab seals the wound and staves off infection. While aninflammatory response brings increased numbers of blood cells to thearea to aid in the repair process, epithelial tissue regenerates andcapillaries grow from blood vessels at the edges of the wound. Thecapillaries revascularize the area of the wound and contribute to theformation of granulation tissue which, in turn, causes scarring.

[0003] Granulation tissue begins to form in the wound site and fills thesite approximately five days after wound induction. Granulation tissuecontains new collagen, fibroblasts, new blood vessels and inflammatorycells, especially macrophages (E. Rubin and J. L. Farber, Pathology,Lippincott, publ., pp. 85-95 (1994)). After seven to ten days, the woundhas regained only 10% of the tissue's original strength.

[0004] Secondary healing causes a greater inflammatory response and moregranulation tissue is formed. In addition, contraction of the wound,resulting from contraction of the fibroblasts of the granulation tissue,brings the edges of the wound together to speed the healing process, butsometimes contributes to disfiguring and debilitating scars.Additionally, excessive deposition of extracellular matrix leads to theformation of keloids, or hypertrophic scars, which areirregularly-shaped, elevated scars that tend toward progressiveenlargement.

[0005] Angiogenesis is generally believed to be a necessary feature ofrepair (Kovacs, E. et al., Fibrogenic cytokines and connective tissueproduction, FASEB J., 8:854-861 (1994). Numerous growth factors andcytokines, secreted first by platelets in response to coagulation andthen by macrophages in response to hypoxia and lactic acidosis,stimulate angiogenesis (Shah, M. et al., The Lancet, 339:213-214(1992)). Angiogenesis generally becomes visible at a microscopic levelabout four days after injury but begins two or three days earlier whennew capillaries sprout out of preexisting venules and grow toward theinjury in response to chemoattractants released by platelets andmacrophages. In primarily closed wounds, sprouting vessels soon meetcounterparts migrating from the other side of the wound and blood flowacross the wound is reestablished. In unclosed wounds, or those not wellclosed, the new capillaries fuse only with neighbors migrating in thesame direction, and a large amount of granulation tissue is formedinstead.

[0006] In normal wound healing, the tissue surrounding a wound undergoesa degree of hypoxia and a concomitant increase in secretion of vascularendothelial growth factor, or VEGF, typically occurring one to two daysfollowing injury (Brown, L. F. et al., Expression of VPF (VEGF) byepidermal keratinocytes during wound healing, J. Exp. Med., 176:1375-79(1992)). VEGF stimulates the rapid proliferation of blood vesselendothelial cells which results in the formation of densely sproutingcapillaries. This rapid hypoxia-induced, VEGF-driven capillary formationstimulates infiltration of inflammatory cells and leads eventually toscarring.

[0007] While inflammation causes scarring, inflammation is alsobeneficial. Inflammatory cells release growth signals and lytic enzymesthat are very important for repair. In fact, patients who receiveanti-inflammatory agents often experience impaired healing due toinadequate inflammation at the site of a wound.

[0008] An important aspect of wound repair is the time involved. Therate at which a wound heals has implications for the prevention ofinfection and improvement of the overall health of the patient. Rapid,even healing without excessive contraction is a desirable result from amedical and cosmetic standpoint.

[0009] Furthermore, it is a recognized clinical phenomenon that surgeryin a tumor patient may lead to tumor progression if the site of thesurgical incision is in proximity to the site of the tumor. In addition,the surgical incisions show high susceptibility to metastaticimplantation. (Murthy et al., Cancer, 64:2035-2044 (1989); Murthy etal., Cancer, 68:1724-1730 (1991); Schackert, H. K. et al., Int. J.Cancer, 44:177-81 (1989)). The stimulatory effect of wounds on tumors ismanifested as accelerated growth of residual tumor near the site ofsurgical intervention, as well as an increased probability of metastaticimplantation at the site of surgery. Furthermore, wounds located at thesite of a tumor regularly fail to heal (Gatenby, R. A. et al.,Suppression of wound healing in tumor bearing animals, Cancer Research,50:7997-8001 (1990)). Persistent wounds that continuously acceleratetumor progression may be a frequent side effect of surgicalinterventions associated with cancer therapy. Therefore, decidingwhether to operate on a tumor patient is often a difficult decision inwhich the benefits of surgery must be compared to the risks of worseninga cancer patient's overall condition.

[0010] It is an object, therefore, of the present invention to provide amethod of preventing or minimizing scar formation during the woundhealing process.

[0011] Another object of the present invention is to provide a method ofaccelerating the rate at which a wound heals.

[0012] A further object of the present invention is to provide a methodof facilitating wound healing in tumor patients and minimizing thelikelihood of tumor progression.

SUMMARY OF THE INVENTION

[0013] The method of the present invention provides for treating apatient having a wound by administering CM101, a generally nontoxicpolysaccharide isolated from group B β-hemolytic Streptococcus (GBS)bacteria, to minimize scarring and to accelerate wound healing. Theinvention finds use in treatment of surface as well as internal wounds.

[0014] Another aspect of the present invention is a method for treatinga keloid by excising the keloid and administering CM101.

[0015] The present invention also provides a method of minimizing thelikelihood of tumor progression, i.e. wound-induced tumor proliferationor metastatic implantation by administration of CM101 before, duringand/or after surgery or other induction of a wound in a tumor patient.

[0016] An article of manufacture including GBS toxin, and particularlyCM101, along with instructions for treatment, and a method of making thearticle are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 graphs vessel density around wounds in tumor-free micetreated with CM101 or saline solution.

[0018]FIG. 2 graphs vessel density around wounds in tumor-bearing micetreated with CM101 or saline solution.

[0019]FIG. 3 presents a graphic representation of the effect of CM101 onthe recovery of skin strength after wounding.

DETAILED DESCRIPTION OF THE INVENTION

[0020] This invention is based in part on the discovery that GBS toxin,and particularly CM101, facilitates wound healing by promoting rapidhealing with minimal scarring. CM101 presumably produces thesebeneficial effects by diminishing the rapid hypoxia-related VEGF-drivenneovascularization that contributes to the scarring associated withwound healing. This mechanism of action also contributes to rapid, evenhealing of wounds in tumor patients, thus reducing the likelihood oftumor proliferation or metastatic implantation at the site of surgery orother wounding.

[0021] CM101, a GBS toxin, is a polysaccharide molecule isolated fromgroup B β-hemolytic Streptococcus (GBS) bacteria. Specifically,pathogenic group B β-hemolytic streptococcus produces a polysaccharideexotoxin. This exotoxin is the putative agent for GBS pneumonia or“early onset disease” in neonatal humans. These newborn infants maysuffer from sepsis, granulocytopenia, and respiratory distress, i.e.pulmonary hypertension and proteinaceous pulmonary edema (Hellerqvist,C. G. et al., Studies on group B β-hemolytic streptococcus I. Isolationand partial characterization of an extra-cellular toxin., Pediatr. Res.,12:892-898 (1981)). It is believed that receptors for CM101 are presentprimarily on the lungs of newborns, making them susceptible to earlyonset disease, but that lung cells lose CM101 receptors approximatelyfour to seven days after birth. Thus, despite the harmful effects onneonates exposed to GBS, CM101 is not known to cause toxicity in olderhumans.

[0022] Isolated CM101 has been shown to have toxic effects on sheepexperimental models that mimic GBS infant pneumonia (Hellerqvist, C. G.et al., Studies on group B β-hemolytic streptococcus I. Isolation andpartial characterization of an extra-cellular toxin., Pediatr. Res.,12:892-898 (1981)). In the sheep model for neonatal early onset disease,GBS toxin causes pulmonary hypertension, increased pulmonary vascularpermeability, granulocytopenia, and pulmonary sequestration ofgranulocytes.

[0023] CM101 has a molecular weight of approximately 300,000 Daltons andcomprises N-acetyl-galactosamine, N-acetyl-glucosamine, glucose,galactose, and mannose residues, in an approximate 1:1:1:3:1 ratio.Carboxylic acid residues are also believed to be integral parts of themolecule. Repeating active epitopes most likely play an important rolein the pathophysiological response to CM101 by crosslinking receptors ontarget endothelium (Hellerqvist, C. G. et al., Early Results of a PhaseI Trial of CM101 in Cancer Patients., Proceedings of the AmericanAssociation of Cancer Research Annual Meeting, 36:224 (1995)).

[0024] A method of preparation of a GBS toxin is provided in U.S. Pat.No. 5,010,062. Preferably, however, the CM101 is purified according tothe method taught in International Application No. PCT/US97/17535,incorporated herein by reference.

[0025] Starting material for isolating CM101 for use in the method ofthe present invention may be obtained by culturing strains of Group Bβ-hemolytic Streptococcus bacteria that have recently infected or arecapable of infecting newborn infants. Isolates of such strains may beobtained from the blood or cerebrospinal fluid of infected infants.

[0026] GBS toxin as used herein is defined as any fraction or componentisolated from natural or lysed GBS bacteria, or derived from mediasupernatants of lysed and/or autoclaved GBS bacteria, and which has abiological activity evidenced by induction of respiratory distress inthe sheep assay (Hellerqvist, C. G. et al., Studies on group Bβ-hemolytic streptococcus I. Isolation and partial characterization ofan extra-cellular toxin., Pediatr. Res., 12:892-898 (1981)) oractivation of complement and binding to neovasculature as demonstratedby a peroxidase-antiperoxidase (PAP) assay of a tumor tissue specimen(Hellerqvist, C. G. et al., Anti-tumor effects of GBS toxin: apolysaccharide exotoxin from group B β-hemolytic streptococcus, J. Canc.Res. Clin. Oncol., 120:63-70 (1993); and Hellerqvist, C. G. et al.,Early Results of a Phase I Trial of CM101 in Cancer Patients.,Proceedings of the American Association of Cancer Research AnnualMeeting, 36:224 (1995)). GBS toxin also means any natural or syntheticpolysaccharide with the same structure or function as any GBS-derivedmolecule with the aforementioned activity.

[0027] Substantially pure GBS toxin means a preparation in which GBStoxin is greater than 40% pure (e.g., present in a concentration of atleast about 40% by weight), preferably at least approximately 60% pure,more preferably at least approximately 90% pure, and most preferably atleast approximately 95% pure. The purity of GBS toxin is discussed ingreater detail in International Application No. PCT/US97/17535. Thedosages described herein are for 95% pure GBS toxin. Dosages of lowerpurity GBS toxin should be altered accordingly.

[0028] One aspect of the present invention is a method of treating apatient having a wound by administering a GBS toxin, e.g. CM101, in anamount sufficient to reduce scarring and/or to accelerate wound healing.Determination of the reduction of scarring and/or the acceleration ofhealing may be performed by a variety of methods including, but notlimited to, visual observation, measurement of vessel density at thesite of the wound, e.g., by magnetic resonance imaging, measurement ofthe amount and/or rate at which granulation tissue is formed, andmeasurement of skin tensile strength at the site of the wound.

[0029] The CM101 or other GBS toxin is preferably combined with apharmaceutically acceptable carrier and administered to a patientsystemically. The carrier is preferably one that is readily mixed withCM101 to form a composition that is administrable by intravenous (IV)means. Thus, the carrier is preferably saline, which may have otherpharmaceutically acceptable excipients included to ensure itssuitability for intravenous administration. The resulting compositionwill be sterile and will have acceptable osmotic properties. In general,a suitable IV formulation is prepared in accordance with standardtechniques known to one of skill in the art. For example, Chapter 85entitled “Intravenous Admixtures” by Salvatore J. Turco in theEighteenth Edition of Remington's Pharmaceutical Sciences, MachPublishing Co. (1990), incorporated herein by reference, providesstandard techniques for preparing a pharmaceutically acceptable IVcomposition useful in accordance with this invention. Other dosage formsto administer CM101 may also be used. As an alternative to systemicadministration, CM101 may be administered locally to a wound site.Administration of CM101 to the patient may occur before, during, and/orafter infliction of a wound by surgery or trauma. Preferably, CM101 isadministered within an appropriate temporal window following thewounding. Administration of CM101 soon after infliction of the wound ismost preferred. For example, administration within 1 day, or preferablywithin six hours is best.

[0030] The amount of CM101 that is administered to a patient to reducescarring or accelerate the rate of wound healing is an amount that issufficient to reduce the amount or rate of granulation tissue formationat a wound site, or that is sufficient to reduce vascularization, andparticularly vessel density, at a wound site. A preferred dosage rangeis 1 to 100 μg/kg body weight. A more preferred dosage range, however,is 1 μg/kg to 50 μg/kg body weight, and most preferred is a dosage inthe range of 1 μg/kg to 25 μg/kg. It will be understood, however, thatthe specific dose level for any particular patient will depend on avariety of factors including the age, body weight, general health, sex,diet, and severity of the wound. Each dosage is preferably administeredin an infusion of up to 120 minutes, with 5 to 60 minutes being thepreferred duration range, and 5 to 30 minutes being the most preferreddosage range. Once weekly treatment is preferred, and is likely to beall that is necessary for evidence of results.

[0031] CM101 treatment inhibits scarring and accelerates healing ofwounds in various types of tissues and is appropriate for wounds ofvarious depths. The treatment of the present invention is preferablyused in conjunction with suturing of muscle tissue or some other knowntissue manipulation for wounds which are deep or in which the edges ofthe tissue are not close together. Scarring at the level of theepidermis as well as internal scarring is reduced by the method of thepresent invention. A scar as used herein is an irregularity of the skinor other tissue formed from connective tissue replacement of tissue,especially tissue damaged by a wound process. An adhesion may also beconsidered a scar in this context. A wound as used herein is injury ordamage wherein the skin or other tissue is adversely affected,particularly wherein the skin or other tissue is torn, pierced, cut, orbroken.

[0032] Additionally, the present invention may be used to treatreperfusion injuries due to surgical wounds or traumatized wounds. Forinstance, blood flow to a part of the body may be temporarily occluded,as by clamping, during a surgical procedure. After removal of theocclusion, reperfusion into a previously ischemic area may result in areperfusion injury to the tissue, especially inside the blood vessel.Reperfusion injury is a major contributor to organ failure and organrejection in transplant surgery. CM101 may be administered to thepatient before occlusion to minimize damage caused by ischemia andreperfusion. CM101 may also be administered after the occlusion hasoccurred. Restenosis, often resulting from scarring within a bloodvessel that leads to stricture of the vessel lumen, is alsoadvantageously treated by the method of the present invention. Further,the invention may be used to minimize the likelihood of formation ofadhesions, such as the type which sometimes occurs as a result ofabdominal surgery. Clearly, the method may be used in specific types ofsurgery, such as plastic or cosmetic surgery, implantation,reconstruction, transplantation, bypass operations, and balloonangioplasty where improved wound healing is critical and scarring isparticularly problematic. Many of these procedures are believed toinvolve hypoxia-induced neovascularization or angiogenesis.

[0033] CM101 is especially useful in the treatment of patients havingkeloids. Normally, surgery to excise these overgrown scars isunsuccessful, because they continually return in a larger and moreunsightly form. The present invention teaches a method of treatmentwhereby surgery to excise the keloid and create a fresh wound isimmediately followed by administration of CM101 to allow for healingwith minimal scarring.

[0034] As stated above, the stimulatory effect of wounds on tumors is arecognized clinical phenomenon that is manifested as accelerated growthof the residual tumor near the site of surgical intervention, as well asan increased probability of metastatic implantation at the site ofsurgery. The present invention also includes administration of CM101 tosurgery patients with tumors in order to facilitate wound healing and tominimize metastatic implantation and tumor proliferation. Treatment forwound healing in surgery patients with tumors includes prevention orreduction of the likelihood of occurrence, and reduction of any tumorthat has moved to the wound site.

[0035] Clearly, administration of CM101 pre-surgery to reduce scarringand accelerate wound healing has great utility and will favorably impactthe medical community. Similarly, the administration of CM101 to apatient having a wound resulting from unexpected injury has greatutility in facilitating healing of the wound.

[0036] Without limitation to a particular theory, it is believed thatGBS toxin, and specifically CM101, plays an important role in allowingwounds to heal at an accelerated rate and with minimal scarring becauseit interferes with the hypoxia-induced, VEGF-driven angiogenesis thatresults in tissue granulation and scarring, but not with physiologicalblood vessel repair processes which are necessary for the wound to heal.Angiogenesis is believed to involve dedifferentiation of endothelialcells in response to upregulation of VEGF under hypoxic conditions,ultimately leading to the rapid formation of densely-sproutingcapillaries. On the other hand, physiological neovascularization isbelieved to be a basic repair mechanism involving proliferation ofexisting endothelial cells after the disruption of contact inhibitionresulting from infliction of a wound. Because CM101 allows physiologicalrepair mechanisms to proceed, but interferes with pathologicalangiogenesis, it is an extremely useful compound for the beneficialtreatment of wounds. The further suggestion is that CM101 opsonizes, bycomplement C3 activation, the budding capillary sprout therebyinhibiting inflammatory angiogenesis necessary for scarring. VEGF-drivenangiogenesis is also believed to be at work in reperfusion injury-typewounds, thus administration of CM101 is effective in preventing suchinjuries.

[0037] Previous work by some of the inventors of the present applicationutilized GBS toxin as an anticancer agent in the treatment of tumors.Particularly, U.S. Pat. No. 5,010,062 to Hellerqvist and thecorresponding European Patent No. EP 0 445 280 B1 teach a method of atleast partially inhibiting vascularization of a developing solid tumorby parenterally administering to a patient GBS toxin in an amounteffective for inhibition.

[0038] The previous work led to investigation of the effect of GBStoxin, and particularly CM101, on wound site vasculature. The conclusionof the early investigation was that CM101 had no effect on woundhealing. Specifically, Quinn, T. E. et al., J. Cancer Res. Clin. Oncol.121:253-6 (1995) utilized a polyvinyl alcohol (PVA) sponge implantationtechnique in mice as a model for wound healing and a carmine dyeinfusion method to measure new vessel formation. In that study, therewas no significant difference in the level of vasculature exhibited bymice treated with CM101 or control Dextran. This was true for bothnormal and tumor-bearing mice. These results indicated that CM101 had nosignificant effects on the neovasculature of healing wounds as measuredby the sponge model. Thus, early studies of the effects of CM101 onwound healing lead one away from the present invention.

[0039] Magnetic resonance imaging (MRI) is useful for visualizingvascularization and providing a relative measure of vessel density. Thistechnique has been used to visualize wound-tumor interactions in vivo innude mice (Abramovitch R., Meir G. and Neeman M., Neovascularizationinduced growth of implanted C6 glioma multicellular spheroids: magneticresonance microimaging, Cancer Res., 55:1956-1962 (1995)) and todemonstrate that wounds influence tumor progression indirectly bystimulating tumor neovascularization and directly by inducing tumor cellproliferation. In the course of these observations, wounds located atthe site of a tumor did not heal.

[0040] Most antiangiogenic or antineovasculature therapies (Broadley. K.N. et al., Lab. Investigation, 61(5):571-575 (1989); Stout, A. J. etal., Int. J. Exp. Pathol., 74(1):79-85 (1993); Pierce, G. F., Annu. Rev.Med., 46:467-481(1995)), with the exception of CM101, inhibit woundhealing and therefore cannot be used to inhibit the stimulatory effectof surgery on tumor growth. It was previously demonstrated that CM101inhibits tumor neovascularization, (U.S. Pat. No. 5,010,062) andprevious investigators concluded that CM101 has no significant effect onneovascularization of wounds (Quinn T. E., Thurman G. B., Sundell A. K.,Zhang M., and Hellerqvist C. G., CM101, a polysaccharide antitumoragent, does not inhibit wound healing in murine models, J. Cancer Res.Clin. Oncol., 121:253-256 (1995)). The present invention, by contrast,teaches that CM101 increases the rate of wound healing, decreasesscarring and further, in cancer patients, acts to inhibit theproliferative effect of wounds on tumors.

[0041] Another aspect of the present invention is an article ofmanufacture, such as a kit, and a method for making the article ofmanufacture. The article includes a pharmaceutical compositioncomprising a GBS toxin, and particularly CM101, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition may be placed in asuitable container, as is well known in the art. Also included areinstructions for treatment of patients according to the methods of thepresent invention.

[0042] The invention now being generally described may be betterunderstood by reference to the following examples, which are presentedfor illustration only and are not to be construed as limitations on thescope or spirit of the present invention.

EXAMPLES Example 1 CM101 Facilitates Wound Healing in Tumor-FreeSubjects

[0043] The effect of CM101 on wound healing was determined in CD-1 nudemice lacking tumors. The wounds were produced by fine surgical scissorsand consisted of 4 mm full thickness skin incisions. A sterile adhesivebandage (Tegaderm™, USA) was used to cover each wound.

[0044] On day 0 of the experiment, mice were intravenously administered240 μg/kg CM101 or saline and wounded. New vessel formation at eachwound site was assessed using magnetic resonance microimaging (MRI).Magnetic resonance images were obtained on days 0, 1, 2, 3, and 5.

[0045] MRI experiments were performed on a horizontal 4.7 T Biospecspectrometer (Bruker, Germany) spectrometer using a 2 cm surfaceradiofrequency coil. Gradient echo images (slice thickness of 0.5 mm, TR100 ms, 256×256 pixels, in plane resolution of 110 μm) were acquiredwith echo times of 10.5 and 20 ms. Growth of the capillary bed wasreflected by reduction of the mean intensity at a region of interest of1 mm surrounding the incision. Angiogenic contrast (AC) was defined asthe ratio of the mean intensity at a region of interest of 1 mmsurrounding the incision to the mean intensity of a distant tissue.Apparent vessel density is given as 1-AC. Each anesthetized mouse wasthen placed supine on an MRI device with the incision site located atthe center of the surface coil, and MRI images were recorded for 1 hour.

[0046]FIG. 1 shows the vessel density measurements by MRI over the timecourse of the experiment. As shown in FIG. 1, wounds in saline-treatedcontrol mice exhibited intense neovascularization near the wound. Thisneovascularization peaks on the second day after injury. In comparison,CM101-treated mice have a severely diminished level ofneovascularization around the wounds.

Example 2 CM101 Facilitates Wound-Healing in Tumor-Bearing Subjects

[0047] The effect of CM101 on neovascularization of wounds was testedusing mice implanted with glioma spheroids. A single C6 glioma spheroid(about 800 μm in diameter) was implanted subcutaneously to the lowerback of each of 6 male CD1-nude mice (Abramovitch R., Meir G. and NeemanM., Neovascularization induced growth of implanted C6 gliomamulticellular spheroids: magnetic resonance microimaging, Cancer Res.,55:1956-1962 (1995)).

[0048] Eight days later, on day 0 of the experiment, the mice wereanesthetized with a intraperitoneal injection of 75 μg/g Ketamine and 3μg/g Xylazine. The mice were injected through the tail vein withphysiological saline containing 0, 60, 120, or 240 μg/kg CM101.Neovascularization at the wound site was assessed using magneticresonance microimaging (MRI), as described in Example 1. Eachanesthetized mouse was placed supine on the MRI device with the tumorlocated at the center of the surface coil, and MRI images were recordedfor one hour.

[0049] After 1 hour of observation and MRI data recording, the mice werewounded by a 4 mm cutaneous incision 5-10 mm from the tumor. As before,the wounds were made through 4 mm full thickness of tissue by finesurgical scissors and were covered with a sterile adhesive bandage(Tegaderm™, USA). Subsequent MRI images of neovascularization were takenon days 2, 5, 7, and 13.

[0050] Wound healing in mice with gliomas was improved by a singleinjection of CM101. Mice treated with CM101 displayed approximatelysix-fold reduced vessel density at the edges of the wound 2 days afterinjury, as seen in FIG. 2. The initial wave of massiveneovascularization following wounding was inhibited in CM101-treatedmice, but this inhibition did not impair wound healing. In fact, at 24and 48 hours after injury, wounds were almost undetectable externally inmice treated with CM101, whereas control mice more clearly showed thewound. Furthermore, compared to saline-treated control mice, CM101treated, tumorbearing mice had almost no detectable scar 13 days afterthe injury.

[0051] Thus, in tumor-bearing subjects treatment with CM101 severelydiminishes the massive neovascularization that surrounds the wound ondays 1-2 after injury, accelerates wound healing, and minimizesscarring.

Example 3 CM101 Treatment Promotes Improved Incision Healing

[0052] Visual observation of wounds also provides evidence of thebenefits of CM101 treatment. Two mice were wounded as in the previousexamples. CM101 was then administered intravenously at a 240 μg/kgdosage in saline to one mouse. Saline was administered to the controlmouse using the same procedure.

[0053] Visual observation of the wound site 48 hours post-incision andpost-CM101 treatment confirmed that the wound site is barely detectable.By contrast, a comparable wound site in the control animal viewed at thesame time point shows clear evidence of wounding. The experimentalanimal also presented little evidence of scarring as compared to thecontrol animal at a later time point.

Example 4 CM101 Accelerates Wound Healing

[0054] Wound disruption strength, or tissue tensile strength across awound, was evaluated with CD1-nude mice. Full thickness skin incisionswere made on the right lumbar region followed by intravenous injectionwith CM101 at 30 and 60 μg/kg or saline. The force needed to disrupt thewound was measured at 40 hours and 7 days after incision. Animalstreated with CM101 demonstrated significant increases relative tocontrols in the strength of the healing wounds.

[0055] Six millimeter incisions were made on day 0. Tensile strength wasmeasured day 2 and day 7 by applying vacuum (Dimensional AnalysisSystems, Inc.) over the wound and registering the movement of two smalldots of reflective material applied on each side of the wound by a videocamera emitting infrared light. FIG. 3 shows that by 2 days, there is ameasurable difference between CM101 treated and control wounds. By 7days, there is no visible scar tissue in the CM101 treated mice and thetissue has the strength of uninjured tissue. In contrast, untreatedwounds were 50% less strong than CM101 treated wounds.

Example 5 CM101 Reduces the Effect of Wound-Induced Tumor Progression

[0056] Twenty-four CD-1 nude mice carrying subcutaneous C6 glioma tumorsof at least 5 mm diameter are prepared.

[0057] The effects of CM101 on tumor proliferation and metastaticimplantation is tested using mice implanted with glioma spheroids. Asingle C6 glioma spheroid (at least 5 mm in diameter) is implantedsubcutaneously to the lower back of each of 24 male CD1-nude mice(Abramovitch R., Meir G. and Neeman M., Neovascularization inducedgrowth of implanted C6 glioma multicellular spheroids: magneticresonance microimaging, Cancer Res., 55:1956-1962 (1995)).

[0058] Eight days later, on day 0 of the experiment, one group of mice(n=8) receive a 4 mm cutaneous incisional injury proximal to the tumor(within 3 mm of the tumor's edge), a second group of mice (n=8) receivea 4 mm cutaneous incisional injury at a location remote from the tumor(more than 10 mm from the tumor's edge), and a control group of mice(n=8) are left without a wound. On the same day, the mice receivetreatments of 0, 60, 120 or 240 μg/kg CM101 (in each group describedabove, two mice receive a particular concentration of CM101).

[0059] For two weeks, tumor growth in situ, tumor implantation at thesite of wound, and neovascularization at the wound site are assessedusing magnetic resonance microimaging (MRI), as described in Example 1.At the end of two weeks, the mice are sacrificed and the tissuesurrounding the tumor and wound are formalin-fixed for histologicalanalysis of wound healing and tumor morphology.

[0060] In animals with wounds proximal to tumors, CM101 treatmentreduces tumor proliferation into the site of the wound. Similarly, CM101inhibits metastatic tumor implantation at the wound site in animals withtumors distant from wounds.

Example 6 CM101 is Effective Against Reperfusion Injury

[0061] The ability of CM101 to guard against reperfusion injury wastested using established animal models (Han et al., Sialyl LewisOligosaccharide Reduces Ischemia-Reperfusion Injury in the Rabbit Ear,J. of Immunol., 155(8):4011-4015 (1995), Missawa et al., Role of SialylLewis ^(x) in Total Hepatic Ischemia and Reperfusion, J. of the AmericanCollege of Surgeons, 182:251-256 (1996), Lopez-Neblina et al., Mechanismof Protection of Verapamil by Preventing Neutrophil Infiltration in theIschemic Rat Kidney, J. Surg. Res., 61(2):469472 (1996). In both the earmodel and the kidney model, the CM101-treated organ exhibitedsignificantly less damage than did the untreated organ.

[0062] Mouse Ear Model: Balb/c mice were injected intravenously witheither phosphate-buffered saline (n=10) or 60 μg/kg CM101 prepared inphosphatebuffered saline (n=10). Following injection, in each mouse, theartery supplying blood to one ear was clamped for a period of 60minutes. At the end of the time period, the clamped ear had lost color,showing the effectiveness of the occlusion. After removal of the clamp,the ear of each mouse was allowed to reperfuse normally. The mice weremaintained at a stable ambient temperature of 24° C. throughout theexperiment. After six hours, the ears of all mice were visually examineddirectly and under a microscope (100×). Measurements of swelling werealso performed using standard calipers.

[0063] The ear that had been subject to clamping was examined in eachsaline-treated mouse and showed swelling and significant vascularleakage indicated by hemorrhage and inflammatory infiltration ascompared to the other ear of the mouse which had not been subject toclamping. By contrast, the reperfused ear of each CM101-treated mouseshowed no significant difference from the unclamped control ear of thesame mouse.

[0064] Mouse Kidney Model: Anesthetized Balb/c mice were opened and theblood flow to one kidney was blocked by clamping. After clamping of theone kidney, either phosphate-buffered saline (Subgroup A) or 60 μg/kgCM101 in phosphate-buffered saline (Subgroup B) was infusedintravenously through the tail vein of each mouse. Five minutes afterthe infusion, the blood flow to the other kidney of each animal wasclamped. Both clamps were removed from each mouse simultaneously afterthe designated occlusion time period and reperfusion was allowed toproceed normally. A stable ambient temperature of 24° C. was maintained.After the designated reperfusion period, each mouse was sacrificed andexamined. The first group of mice {6 saline-treated mice (Group 1A) and6 CM101-treated mice (Group 1B)} had the blood flow to their kidneysoccluded by clamping for 30 minutes and reperfusion was allowed toproceed for 2 hours. The second group {6 saline-treated mice (Group 2A)and 6 CM101-treated mice (Group 2B)} were subject to a 45 minuteocclusion period and a 5 hour reperfusion period. The third group {6saline-treated mice (Group 3A) and 6 CM101-treated mice (Group 3B)} wassubject to occlusion for 60 minutes followed by reperfusion for 6 hours.

[0065] In the saline-treated mice, histological examination of thesaline-treated kidneys revealed signs of reperfusion damage, showinggreater injury with increased reperfusion time. Thus, damage tocapillaries was evident after 2 hours of reperfusion (Group 1A) andevidence of angiogenesis, hemorrhage, and inflammation was observed inthe 6-hour reperfusion mice (Group 3A). Additionally, cell death wasobserved by 4 hours (Group 2A). By contrast, at all time points theCM101-treated mice showed minor dilation of blood vessels, but nohemorrhaging between blood vessels, inflammation, or cell death. Thisdemonstrates the protective role of CM101 in ischemia and reperfusioninjury.

[0066] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0067] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

What is claimed is:
 1. A method for treating a patient having a wound,which method comprises: administering a group B β-hemolyticStreptococcus (GBS) toxin to the patient in a quantity sufficient toreduce scarring at a site of the wound.
 2. A method of treating apatient having a wound, which method comprises: administering a group Bβ-hemolytic Streptococcus (GBS) toxin to the patient in an amountsufficient to accelerate wound healing.
 3. The method of claim 1 whereinhealing of the wound is accelerated.
 4. The method of claim 2 where thewound healing is achieved with minimal scarring.
 5. The method of claim1 or 2 wherein the GBS toxin is CM101.
 6. The method of claim 5 whereinthe CM101 is substantially pure.
 7. The method of claim 6 wherein theCM101 has a purity of at least approximately 90%.
 8. The method of claim1 or 2 wherein the GBS toxin is administered to the patient in aquantity sufficient to provide reduced vessel density at the site of thewound with GBS toxin treatment relative to vessel density at the site ofa comparable wound without GBS treatment.
 9. The method of claim 8wherein the reduced vessel density is within the range of 0.0 to 0.2(1-AC).
 10. The method of claim 1 or 2 wherein the GBS toxin isadministered to the patient in a quantity sufficient to provide improvedtensile strength at the site of the wound with GBS toxin treatmentrelative to tensile strength at the site of the wound without GBStreatment.
 11. The method of claim 1 or 2 wherein the GBS toxin isadministered to the patient parenterally.
 12. The method of claim 11wherein the GBS toxin is administered to the patient intravenously. 13.The method of claim 1 or 2 wherein the GBS toxin is administeredproximate to the site of the wound.
 14. The method of claim 1 or 2wherein the GBS toxin is administered to the patient at a dosage in therange of 1 to 100 μg/kg body weight.
 15. The method of claim 14 whereinthe GBS toxin is administered to the patient at a dosage in the range of1 to 25 μg/kg body weight.
 16. The method of claim 14 wherein the dosageis administered in a single infusion of thirty minute duration or less.17. The method of claim 14 wherein the dosage is administered onceweekly.
 18. The method of claim 1 or 2 wherein the GBS toxin isadministered to the patient in a quantity sufficient to reducevascularization at the site of the wound.
 19. The method of claim 18wherein the GBS toxin is administered to the patient in a quantitysufficient to reduce hypoxia-induced, VEGF-driven neovascularization atthe site of the wound.
 20. The method of claim 1 or 2 wherein thepatient has a tumor.
 20. 21. The method of claim 1 or 2 wherein thepatient has no known tumor.
 22. The method of claim 1 or 2 wherein thepatient is at least four days old.
 23. The method of claim 22 whereinthe patient is at least seven days old.
 24. The method of claim 1 or 2wherein the wound is the result of trauma.
 25. The method of claim 1 or2 wherein the wound is the result of surgery.
 26. The method of claim 1or 2 wherein the wound is within a blood vessel.
 27. A method oftreating a patient having a keloid, which method comprises: (a) excisingthe keloid to create a wound, and (b) administering a group Bβ-hemolytic Streptococcus (GBS) toxin to the patient in a quantitysufficient to reduce scarring at a site of the wound.
 28. The method ofclaim 27 wherein the GBS toxin is CM101.
 29. The method of claim 28wherein the CM101 has a purity of at least approximately 90%.
 30. Amethod of reducing the likelihood of metastatic tumor implantationproximate to a site of surgery in a surgery patient, which methodcomprises: administering a group B β-hemolytic Streptococcus (GBS) toxinto the surgery patient in a quantity sufficient to reduce scarring oraccelerate wound healing at the site of surgery.
 31. The method of claim30 wherein the GBS toxin is CM101.
 32. The method of claim 31 whereinthe CM101 has a purity of at least approximately 90%.
 33. The method ofclaim 30 wherein the GBS toxin is administered to the patientintravenously.
 34. A method of reducing the likelihood of tumorproliferation in a surgery patient, which method comprises:administering a group B β-hemolytic Streptococcus (GBS) toxin to thesurgery patient in a quantity sufficient to reduce scarring oraccelerate wound healing at a site of surgery.
 35. The method of claim34 wherein the GBS toxin is CM101.
 36. The method of claim 35 whereinthe CM101 has a purity of at least approximately 90%.
 37. The method ofclaim 34 wherein the GBS toxin is administered to the patientintravenously.
 38. A method of protecting against reperfusion injury ina patient, which method comprises: administering a group B β-hemolyticStreptococcus (GBS) toxin to the patient prior to reperfusion of a bloodvessel.
 39. The method of claim 38 wherein the GBS toxin is administeredprior to occlusion of the blood vessel.
 40. An article of manufacturecomprising: (a) a pharmaceutical composition having (i) a group Bβ-hemolytic Streptococcus (GBS) toxin, and (ii) a pharmaceuticallyacceptable carrier, and (b) instructions for administering thepharmaceutical composition to a patient having a wound.
 41. The articleof claim 40 wherein the GBS toxin is CM101.
 42. The article of claim 41wherein the CM101 has a purity of at least approximately 90%.
 43. Thearticle of claim 40 wherein the instructions describe administration ofthe pharmaceutical composition to the patient in a quantity sufficientto reduce scarring at a site of the wound.
 44. The article of claim 40wherein the instructions describe administration of the pharmaceuticalcomposition to the patient in a quantity sufficient to accelerate woundhealing.
 45. The article of claim 40 wherein the instructions describeadministration of the pharmaceutical composition to the patient to treata keloid by excising the keloid and administering the pharmaceuticalcomposition in a quantity sufficient to reduce scarring at a site of thewound.
 46. The article of claim 40 wherein the instructions describeadministration of the pharmaceutical composition to the patient toreduce the likelihood of metastatic tumor implantation proximate to asite of surgery in the patient by administering the pharmaceuticalcomposition in a quantity sufficient to reduce scarring or acceleratewound healing at the site of surgery.
 47. The article of claim 40wherein the instructions describe administration of the pharmaceuticalcomposition to the patient to reduce the likelihood of tumorproliferation in the patient by administering the pharmaceuticalcomposition in a quantity sufficient to reduce scarring or acceleratewound healing at a site of surgery.
 48. A method of making an article ofmanufacture, which method comprises: combining (a) a container includinga pharmaceutical composition comprising (i) a group B β-hemolyticStreptococcus (GBS) toxin, and (ii) a pharmaceutically acceptablecarrier, and (b) labelling instructions for treating a patient having awound by administering the pharmaceutical composition to the patient.49. The method of claim 48 wherein the instructions describeadministration of the pharmaceutical composition to the patient in aquantity sufficient to reduce scarring at a site of the wound.
 50. Themethod of claim 48 wherein the instructions describe administration ofthe pharmaceutical composition to the patient in a quantity sufficientto accelerate wound healing.
 51. The method of claim 48 wherein theinstructions describe administration of the pharmaceutical compositionto the patient to treat a keloid by excising the keloid andadministrating the pharmaceutical composition in a quantity sufficientto reduce scarring at a site of the wound.
 52. The method of claim 48wherein the instructions describe administration of the pharmaceuticalcomposition to the patient to reduce the likelihood of metastatic tumorimplantation proximate to a site of surgery in the patient byadministering the pharmaceutical composition in a quantity sufficient toreduce scarring or accelerate wound healing at the site of surgery. 53.The method of claim 48 wherein the instructions describe administrationof the pharmaceutical composition to the patient to reduce thelikelihood of tumor proliferation in the patient by administering thepharmaceutical composition in a quantity sufficient to reduce scarringor accelerate wound healing at a site of surgery.